Method for treating fibromyalgia and chronic fatigue syndrome

ABSTRACT

The invention provides methods and compositions for treating fibromyalgia (FM) and chronic fatigue syndrome (CFS) in an individual. The methods provided herein entail administering a composition comprising an isolated Mycobacterium or antigenic fragments derived therefrom. Also provided herein are methods for assessing alleviation of symptoms and/or alteration of immune system functioning following administration of a composition comprising an isolated Mycobacterium or antigenic fragments derived therefrom.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/055,565, filed Aug. 6, 2018, now U.S. Pat. No. 11,077,179, whichclaims benefit from U.S. Provisional Application No. 62/543,478, filedAug. 10, 2017, and U.S. Provisional Application No. 62/642,621, filedMar. 14, 2018, each of which is incorporated by reference herein in itsentirety for all purposes.

FIELD OF THE INVENTION

This invention provides methods for treating fibromyalgia and chronicfatigue syndrome (CFS) using compositions comprising isolatedMycobacteria or antigenic fragments derived therefrom. Provided hereinare also methods for diagnosing CFS by measuring levels of one or moreselect cytokines.

BACKGROUND OF THE INVENTION

Fibromyalgia (FM) or FM syndrome is a medical disorder of unknownetiology characterized by chronic widespread joint and muscle pain.Other symptoms include allodynia (a heightened and painful response topressure), debilitating fatigue, sleep disturbance, and joint stiffness,numbness, tingling and cognitive dysfunction. The etio-pathophysiologyof FM is not entirely understood, although inflammation in themusculoskeletal system of FM patients shows that the immune system playsa role in the etio-pathophysiology of FM. In addition, genetic andenvironmental factors have been suggested to play a role in theetio-pathophysiology of FM.

This debilitating, chronic affliction affects 10 million Americans andthere is no known cure for the disease. Although fibromyalgia does nothave a specific treatment or treatment paradigm, three medications havebeen approved by the FDA for the treatment of fibromyalgia. These arepregabalin (Lyrica®), duloxetine (Cymbalta®), and milnacipran(Savella®). However, recent research indicates that current treatmentsare really not effective in the reduction of pain or improvement infunction in patients with fibromyalgia, and there is still a lack ofeffective drugs for the treatment of fibromyalgia over time (Wolfe etal., 2012, “Longitudinal Patterns of Analgesic and Central Acting DrugUse and Associated Effectiveness in Fibromyalgia,” Eur. J. Pain 17(4):581-86).

Accordingly, there is a need for a treatment of FM that has long lastingeffects on a majority or all of the symptoms associated with FM. Thepresent invention addresses this and other needs.

Chronic fatigue syndrome (CFS) (also referred to as myalgicencephalomyelitis (ME) or systemic intolerance disease (SEID)) is aserious, debilitating condition that affects somewhere between 836,000and 2.5 million Americans. CFS is a clinically defined conditioncharacterized by severe disabling flu-like fatigue following exertion ofany sort (physical, cognitive or emotional) as well as a combination ofsymptoms that include reduction or impairment in ability to carry outnormal daily activities, cognitive impairment (e.g., impairment ofconcentration and short-term memory), sleep disturbances (unrefreshingsleep), and orthostatic intolerance (symptoms that worsen when a personstands upright and improve when the person lies back down). Other commonmanifestations include musculoskeletal pain, failure to recover from aprior infection, abnormal immune function. Further, symptoms can persistfor years with one-quarter of ME/CFS patients being bed or house boundat some point in their illness and most patients never regaining theirpre-disease level of health or functioning. Overall, ME/CFS patients canexperience a loss of productivity as well as high medical costs thatcontribute to a total economic burden of $17 to $24 billion annually.

Currently, the cause of ME/CFS remains unknown, although there is someevidence that symptoms may be triggered by certain infections. Further,no specific test exists to diagnose chronic fatigue syndrome and thereis no existing cure for ME/CFS. While there are therapies available tomanage symptoms, the efficacy of said therapies is largely unknown.

Accordingly, there is a need for a treatment of CFS that has longlasting effects on a majority or all of the symptoms associated withCFS. The present invention addresses this and other needs.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a method of treating or preventingfibromyalgia in a subject, comprising administering a therapeuticallyeffective amount of a vaccine to the subject, wherein the vaccinecomprises Mycobacterium bovis or an antigenic fragment thereof. In somecases, the M. bovis is live-attenuated. In some cases, the M. bovis isheat-killed. In some cases, the M. bovis is a Bacille Calmette-Guerin(BCG) strain. In some cases, the BCG strain belongs to duplication groupDU1, DU2-I, DU2-II, DU2-III or DU2-IV. In some cases, the BCG strain isa sub-strain selected from BCG Pasteur (1961), BCG Moreau, BCG Tokyo,BCG Russia (1924), BCG Japan (1925), BCG Sweden (1927), BCG Birkhaug,BCG Prague, BCG Glaxo (1954), BCG Merieux (1989), BCG Danish, BCGFrappier, BCG Connaught (1948), BCG Mexico, BCG Tice (1934), BCG Chinaor BCG Phipps. In some cases, the BCG strain is BCG Tokyo. In somecases, the vaccine further comprises an adjuvant. In some cases, thevaccine induces one or more epigenetic changes in the genome of thesubject. In some cases, the vaccine is administered in repeat doses. Insome cases, the vaccine is administered in a unit dose comprising1.8×10⁶ to 3.9×10⁶ colony forming units/injection of 0.1 ml. In somecases, the vaccine is formulated for intradermal administration. In somecases, the administration of the vaccine alleviates, prevents or reducesat least one sign or symptom of fibromyalgia, wherein the sign orsymptom is selected from chronic muscle pain, muscle spasms, muscletightness, moderate or severe fatigue, decreased energy, insomnia,feeling of exhaustion, stiffness upon waking, stiffness after staying inone position for too long, difficulty remembering, difficultyconcentrating, difficulty performing simple mental tasks (“fibro fog”),abdominal pain, bloating, nausea, constipation alternating with diarrhea(irritable bowel syndrome), tension or migraine headaches, jaw andfacial tenderness, sensitivity to one or more of odors, noise, brightlights, medications, certain foods, and cold, feeling anxious ordepressed, numbness or tingling in the face, arms, hands, legs, or feet,increase in urinary urgency or frequency (irritable bladder), reducedtolerance for exercise and muscle pain after exercise. a feeling ofswelling (without actual swelling) in the hands and feet or anycombination of symptoms thereof. In some cases, the administration ofthe vaccine elevates or increases immune system activity of the subject.In some cases, the elevation or increase in immune system function isevidenced by the production of T_(H)1 cytokines, upregulation ofgranzyme B or both.

In another aspect, provided herein is a method of elevating orincreasing a subject's immune system function comprising administering atherapeutically effective amount of a vaccine to the subject, whereinthe subject suffers from or is suspected of suffering from fibromyalgiaand wherein the vaccine comprises Mycobacterium bovis or an antigenicfragment thereof. In some cases, the elevation or increase in immunesystem function is evidenced by the production of T_(H)1 cytokines,upregulation of granzyme B or both. In some cases, the elevation orincrease in immune system function is evidenced by a reduction oralleviation of at least one sign or symptom of fibromyalgia. In somecases, the sign or symptom of fibromyalgia is selected from chronicmuscle pain, muscle spasms, muscle tightness, moderate or severefatigue, decreased energy, insomnia, a feeling of exhaustion, stiffnessupon waking, stiffness after staying in one position for too long,difficulty remembering, difficulty concentrating, difficulty performingsimple mental tasks (“fibro fog”), abdominal pain, bloating, nausea, andconstipation alternating with diarrhea (irritable bowel syndrome),tension or migraine headaches, jaw and facial tenderness, sensitivity toone or more of odors, noise, bright lights, medications, certain foods,and cold, feeling anxious or depressed, numbness or tingling in theface, arms, hands, legs, or feet, increase in urinary urgency orfrequency (irritable bladder), reduced tolerance for exercise and musclepain after exercise. a feeling of swelling (without actual swelling) inthe hands and feet or a combination of symptoms thereof. In some cases,the vaccine induces one or more epigenetic changes in the genome of theindividual. In some cases, the vaccine is live-attenuated. In somecases, the vaccine is heat-killed. In some cases, the M. bovis is aBacille Calmette-Guerin (BCG) strain. In some cases, the BCG strainbelongs to duplication group DU1, DU2-I, DU2-II, DU2-III or DU2-IV. Insome cases, the BCG strain is a sub-strain selected from BCG Pasteur(1961), BCG Moreau, BCG Tokyo, BCG Russia (1924), BCG Japan (1925), BCGSweden (1927), BCG Birkhaug, BCG Prague, BCG Glaxo (1954), BCG Merieux(1989), BCG Danish, BCG Frappier, BCG Connaught (1948), BCG Mexico, BCGTice (1934), BCG China or BCG Phipps. In some cases, the BCG strain isBCG Tokyo. In some cases, the vaccine further comprises an adjuvant. Insome cases, the vaccine is administered in repeat doses. In some cases,the vaccine is administered in a unit dose comprising 1.8×10⁶ to 3.9×10⁶colony forming units/injection of 0.1 ml. In some cases, the vaccine isformulated for intradermal administration.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” refers to one or more of that entity, i.e. canrefer to a plural referents. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (3^(rd) ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The term “cytokine” as used herein refers to small proteins that aresecreted by specific cells of the immune system and glial cells, andinclude lymphokines, interleukins, and chemokines and theircorresponding receptors, such as but not limited to IL-1, IL-2, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-21, IFN-γ,IFN-α, TNF-α, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, GM-CSF, Eotaxin,RANTES, etc. In another aspect, the invention further includesdetermining the levels of one or more of IL-1RA, IL2R, IL-7, IL-12(p40/p70), IL-13, IL-15, IL-17, IFN-α, IP-10, MIG, VEGF, G-CSF, EGF,FGF-basic and HGF. In yet another aspect, the invention also includesdetermining the levels of IL-9 and PDGF-BB or a combination thereof. Thecytokine may be inflammatory or anti-inflammatory. In one embodiment,the cytokine to be assayed may be a full length polypeptide, protein, aglycoprotein or a fragment thereof. Other proteins that can be assayedinclude hormones, heat-shock proteins, antibodies such as but notlimited to anti-nuclear antibody (ANA), thyroid antibodies,anti-extractable nuclear antibodies (ENA), IgG subclasses, anti-nuclearfactors (FAN), rheumatoid factor (RF), receptor proteins and ligands,etc. In other embodiment, the level of cytokine assayed maybe a mRNA,miRNA, or DNA.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent described herein, or identified bya method described herein, to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.

The terms “patient”, “subject” and “individual” are used interchangeablyherein, and mean a mammalian subject to be treated, with human patientsbeing preferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary applications, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters, as well as primates.

By the phrases “therapeutically effective amount” and “effective dosage”is meant an amount sufficient to produce a therapeutically (e.g.,clinically) desirable result; the exact nature of the result will varydepending on the nature of the disorder being treated. For example,where the disorder to be treated is fibromyalgia (FM), the result can bean alleviation of one or more symptoms of FM such as, for example,widespread pain. The skilled artisan will appreciate that certainfactors can influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of the compositions ofthe invention can include a single treatment or a series of treatments.

Overview

The present invention provides compositions and methods for treatingfibromyalgia (FM) in an individual. In one embodiment, treating FM in anindividual comprises administering a vaccine. The vaccine can be anyvaccine that can induce epigenetic changes in an individual administeredsaid vaccine. The epigenetic changes can serve to provide the individualwith life-long immunity against re-occurrence of FM. The epigeneticchanges can be cis-acting or trans-acting. The epigenetic changes caninclude changes in DNA methylation and/or histone protein modification.The vaccine can induce a chemokine and/or cytokine response in theindividual. The epigenetic changes or modifications can impact thechemokine and/or cytokine responses in the individual. The vaccinecomposition can comprise a Mycobacterium or an antigenic fragmentthereof. The Mycobacterium can be an isolated Mycobacterium or anantigenic fragment thereof. In one embodiment, the Mycobacterium is aBacille Calmette-Guerin (BCG) strain of Mycobacterium bovis (M. bovis).Further to this embodiment, the composition can be a BCG vaccine. TheBCG vaccine can be any BCG vaccine known in the art and/or commerciallyavailable. In a preferred embodiment, the BCG vaccine comprises theTokyo 172 strain of BCG (e.g., Type I or Type II). The BCG vaccine canbe live-attenuated or heat-killed. In another embodiment, theMycobacterium is a non-pathogenic Mycobacterium species such as, forexample Mycobacterium vaccae or Mycobacterium obtuense. Thenon-pathogenic Mycobacterium can be live-attenuated or heat-killed. Theindividual may have been previously diagnosed with FM or may besuspected of suffering from or being afflicted with FM. In oneembodiment, the individual was previously diagnosed as having FM usingany diagnostic means or methods known in the art such as, for example,the FM/a® Fibromyalgia test. In another embodiment, the individual waspreviously diagnosed as having FM using the methods disclosed inUS20150301062A1, the contents of which are herein incorporated byreference in their entirety.

In one embodiment, the FM diagnosis can be determined by detecting thelevels of one or more cytokines in a sample obtained from the individualto see if the levels of the one or more cytokines are altered. Furtherto this example, to determine whether cytokine levels are altered, thecytokine levels in the individual are compared to control cytokinelevels, for example, cytokine levels from a healthy patient, or cytokinelevels reported for a patient without fibromyalgia (for example, levelsreported in a database). In one embodiment, a positive diagnosis offibromyalgia is provided if a majority of the cytokines tested havealtered expression. In a further embodiment, a positive diagnosis offibromyalgia is provided if at least about 67% of the cytokines testedhave altered expression, or at least about 67% or more of the cytokinestested have altered expression. In a further embodiment, a positivediagnosis of fibromyalgia is provided if at least about 75%, or at leastabout 75% or more of the cytokines tested have altered expression. Ineven a further embodiment, a positive diagnosis of fibromyalgia isprovided if the expression level of every cytokine tested, or aboutevery cytokine tested in the patient is altered. In one embodiment,altered expression is determined by comparing the cytokine levels of theindividual's sample to control levels. Control levels, in oneembodiment, are determined by testing a sample of an individual known tonot have FM. In another embodiment, control levels are known, forexample, from a database. The altered level(s) of the cytokines measuredin the affected individual compared to the level from control group ispredictive/indicative of FM in the individual. The cytokine levels in anindividual with FM, for example, cytokine levels in a FM patient'sblood, in one embodiment, are higher than the cytokine levels of ahealthy patient, for each cytokine tested. In another embodiment, thecytokine levels in a FM patient's blood are lower than the cytokinelevels of a healthy patient, for each cytokine tested. In yet anotherembodiment, the cytokine levels measured in a patient with FM may behigher or lower, depending on the panel of cytokines measured in theindividual.

In one embodiment, the FM diagnosis can be determined by evaluating anindividual suspected of suffering from FM with the FibroFatigue scale.The FibroFatigue scale is an observer's rating scale with 12 itemsmeasuring pain, muscular tension, fatigue, concentration difficulties,failing memory, irritability, sadness, sleep disturbances, and autonomicdisturbances (items derived from the CPRS) and irritable bowel,headache, and subjective experience of infection as described inZachrisson et al., J Psychosom Res. 2002 June; 52(6):501-9, which isherein incorporated by reference in its entirety. The FibroFatigue scalecan be conducted by a trained administrator. In another embodiment, theFM diagnosis can be determined by evaluating an individual suspected ofsuffering from FM with the FibroFatigue Scale in combination withdetecting the levels of one or more cytokines in a sample obtained fromthe individual to see if the levels of the one or more cytokines arealtered as described herein.

The present invention also provides compositions and methods fortreating chronic fatigue syndrome (CFS) in an individual. In oneembodiment, treating CFS in an individual comprises administering avaccine. The vaccine can be any vaccine that can induce epigeneticchanges in an individual administered said vaccine. The epigeneticchanges can serve to provide the individual with life-long immunityagainst re-occurrence of a disease or condition such as CFS. Theepigenetic changes can be cis-acting or trans-acting. The epigeneticchanges can include changes in DNA methylation and/or histone proteinmodification. The vaccine can induce a chemokine and/or cytokineresponse in the individual. The epigenetic changes or modifications canimpact the chemokine and/or cytokine responses in the individual. Thevaccine composition comprises a Mycobacterium or an antigenic fragmentthereof. The Mycobacterium can be an isolated Mycobacterium or anantigenic fragment thereof. In one embodiment, the Mycobacterium is aBacille Calmette-Guerin (BCG) strain of Mycobacterium bovis (M. bovis).Further to this embodiment, the composition can be a BCG vaccine. TheBCG vaccine can be any BCG vaccine known in the art and/or commerciallyavailable. In a preferred embodiment, the BCG vaccine comprises theTokyo 172 strain of BCG (e.g., Type I or Type II). The BCG vaccine canbe live-attenuated or heat-killed. In another embodiment, theMycobacterium is a non-pathogenic Mycobacterium species such as, forexample Mycobacterium vaccae or Mycobacterium obtuense. Thenon-pathogenic Mycobacterium can be live-attenuated or heat-killed. Theindividual may have been previously diagnosed with CFS or may besuspected of suffering from or being afflicted with CFS. In oneembodiment, the individual was previously diagnosed as having CFS usingany diagnostic means or methods known in the art such as, for example,following an appropriate history, physical examination, and medicalwork-up. In one embodiment, the individual was previously diagnosed ashaving CFS using the commercially available FM/a® Fibromyalgia test. Inanother embodiment, the individual was previously diagnosed as havingCFS using the methods disclosed in US20150301062A1, the contents ofwhich are herein incorporated by reference in their entirety.

The CFS diagnosis can be determined following assessment of a subject'shistory, physical examination by a medical professional (e.g.,physician) and a medical work-up. The CFS diagnosis can be assigned ordetermined if a subject experiences a specific set of symptoms that canbe each of: 1.) a substantial reduction or impairment in the ability toengage in pre-illness levels of occupational, educational, social, orpersonal activities that persists for more than 6 months and isaccompanied by fatigue, which is often profound, is of new or definiteonset (not lifelong), is not the result of ongoing excessive exertion,and is not substantially alleviated by rest; 2.) post-exertionalmalaise; and 3.) unrefreshing sleep. A CFS diagnosis can be assigned ifthe subject experiences each of the aforementioned set of symptoms incombination with one or both of cognitive impairment and orthostaticintolerance. A positive CFS diagnosis can be ascribed to a subject ifthe subject experiences the aforementioned set of symptoms alone or incombination with cognitive impairment and/or orthostatic intolerance forat least 6 months. A positive CFS diagnosis can be ascribed to a subjectif the subject experiences the aforementioned set of symptoms alone orin combination with cognitive impairment and/or orthostatic intolerancefor at least 6 months and can be described as being chronic, frequentand/or moderate to severe in intensity. Other symptoms that a subjectmay experience can be pain, certain infectious diseases,gastrointestinal and genitourinary problems, sore or scratchy throat,painful or tender axillary/cervical lymph nodes, sensitivity to externalstimuli, immune system problems, hormonal imbalances or any combinationthereof. The certain infectious diseases can be viral infections withthe Epstein-Barr virus, human herpes virus 6 (HHV-6), coxsackievirus B,spumaviruses, human T-cell leukemia virus strains, and/or mouseretroviruses. The certain infectious diseases can be pneumonia, diarrheaor upper respiratory tract infections. In some cases, diagnosis of CFSin a subject can comprise determining the presence and/or level ofantibodies (e.g., IgGs) to antigens derived from one or more of theaforementioned viruses and/or Chlamydia pneumoniae.

In another embodiment, the CFS diagnosis can be determined by detectingthe levels of one or more cytokines in a sample obtained from anindividual to see if the levels of the one or more cytokines arealtered. In one embodiment, the method comprises determining whether thelevels of at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve or at least thirteen cytokines, or atleast thirteen or more cytokines in an individual are altered. In oneembodiment, the methods for diagnosing or predicting CFS in a patientinvolve determining or assaying the levels of at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, or at least ten or morecytokines in the plasma of blood samples obtained from individualssuspected of being afflicted with CFS or at risk for CFS. In a furtherembodiment, the method involves determining or assaying the levels of atleast one, at least two, at least three, at least four, at least five,at least six, at least seven, at least eight, at least nine, or at leastten or more cytokines in the peripheral blood mononuclear cells (PBMCs)that have been separated from the plasma of blood samples obtained fromthe individuals. These levels are then analyzed to determine if thelevels are altered. The alteration may be an increase or decrease inexpression of a cytokine. The alteration can be determined at theprotein and/or mRNA level as provided herein.

In one embodiment, altered expression is determined by comparing thecytokine levels of the individual's sample to control levels. Controllevels, in one embodiment, are determined by testing a sample from ahealthy individual or an individual known to not have CFS. In anotherembodiment, control levels are known, for example, from a database. Thealtered level(s) of the cytokines measured in the affected individualcompared to the level from the control group is predictive/indicative ofCFS in the individual. In one embodiment, a positive diagnosis of CFS isprovided if at least about 33% of the cytokines tested have alteredexpression, or at least about 33% or more of the cytokines tested havealtered expression. In another embodiment, a positive diagnosis of CFSis provided if a majority of the cytokines tested have alteredexpression. In a further embodiment, a positive diagnosis of CFS isprovided if at least about 67% of the cytokines tested have alteredexpression, or at least about 67% or more of the cytokines tested havealtered expression. In a further embodiment, a positive diagnosis of CFSis provided if at least about 75%, or at least about 75% or more of thecytokines tested have altered expression. In even a further embodiment,a positive diagnosis of CFS is provided if the expression level of everycytokine tested, or about every cytokine tested in the patient isaltered. The sample can be a solid sample (e.g., tissue biopsy) or aliquid sample (e.g., blood sample or a fraction thereof). As describedherein, altered expression can be an increase or decrease in expression.The altered expression can be at the protein and/or mRNA level asdescribed herein.

The cytokine levels in an individual with CFS, for example, cytokinelevels in a CFS patient's blood, in one embodiment, are higher than thecytokine levels of a healthy patient, for each cytokine tested. Inanother embodiment, the cytokine levels in a CFS patient's blood arelower than the cytokine levels of a healthy patient, for each cytokinetested. In yet another embodiment, the cytokine levels measured in apatient with CFS may be higher or lower, depending on the panel ofcytokines measured in the individual.

The present invention is not limited by any particular combination ofcytokines. For example, the expression levels of cytokines included incommercial cytokine panels (or cytokine subsets thereof) can beevaluated by the methods provided herein. Various combinations ofcytokines for use in the present invention are provided in Table 1below. Subsets of these combinations may also be used in the methodsprovided herein. It should be understood that these combinations arerepresentative, and should not be construed as limiting the invention.

TABLE 1 Non-limiting cytokine panels for use with the present invention.Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 IL5 IFN-γ TranSignal Bio-PlexPro five or more IL6 IL-1β Human Cytokine magnetic chemokines IL8 IL-2Antibody Array Cytokine Assay IL 10 IL-4 3.0 (any of these IFN-γ IL-5(or a subset of assays may be MCP-1 IL-6 cytokines used, i.e., theMIP-1α IL-8 provided in this 8-plex, 17-plex, MIP-1β IL-10 assay)21-plex, TNF-α 27-plex MIP-1β MCP-1 MIP-1α Rantes Panel 6 Panel 7 Panel8 Panel 9 Panel 10 IL5 IL-6 IL-2 IL-8 IFN-γ IL6 IL-8 IL-4 IL-10 IL-1βIL8 IL-10 IL-5 TNF-α IL-2 IL 10 TNF-α IL-6 MIP-1β IFN-γ MIP-1β MCP-1MCP-1 Rantes MIP-1α

The present invention also provides compositions and methods fortreating an individual suffering from or suspected of suffering fromconditions that share many clinical illness features with FM and/or CFSor any comorbid illnesses. An example of conditions that shares clinicalillness features of myalgia, fatigue, sleep disturbances and impairmentin ability to perform activities of daily living is temporomandibulardisorder (TMD). Examples of illnesses comorbid with FM and/or CFS can beirritable bowel sydrome (IBS), chronic tension-type headache andinterstitial cystitis. The treatment can be a vaccine as providedherein.

In one embodiment, administration, as defined herein, includes theadministration of the vaccine (e.g., Mycobacterium or an antigenicfragment thereof) in multiple aliquots and/or doses and/or on separateoccasions. The vaccine can be a Mycobacterium vaccine. The Mycobacteriumvaccine can comprise a Mycobacterium or an antigenic fragment thereof.

In one aspect of the present invention, the Mycobacterium comprises alive-attenuated strain of a Mycobacterial species or an antigenicfragment thereof. In another aspect of the present invention theMycobacterium comprises heat-killed strain of a Mycobacterial species oran antigenic fragment thereof.

Mycobacterial species for use in the present invention include, but arenot limited to M. vaccae, M. thermoresistibile, M. flavescens, M.duvalii, M. phlei, M. obuense, M. parafortuitum, M. sphagni, M.aichiense, M. rhodesiae, M. neoaurum, M. chubuense, M. tokaiense, M.komossense, M. aurum, M. indicus pranii, M. tuberculosis, M. micron; M.africanum; M. kansasii, M. marinum; M. simiae; M. gastri; M.nonchromogenicum; M. terrae; M. triviale; M. gordonae; M. scrofulaceum;M. paraffinicum; M. intracellulare; M. avium; M. xenopi; M. ulcerans; M.diemhoferi, M. smegmatis; M. thamnopheos; M. flavescens; M. fortuitum;M. peregrinum; M. chelonei; M. paratuberculosis; M. leprae; M.lepraemurium; M. bovis and combinations thereof.

In one embodiment, the Mycobacterial species is M. bovis. The M. boviscan be any known strain of M. bovis. In one embodiment, the M. bovis isa BCG strain. The BCG can be heat-killed or live-attenuated. The BCG canbe any known BCG vaccine. The strain of BCG can be any of the strainsbelonging to BCG groups DU1, DU2-I, DU2-II, DU2-III and DU2-IV. Thestrain of BCG can be selected from BCG Pasteur (1961), BCG Moreau, BCGRussia (1924), BCG Japan (1925), BCG Tokyo 172 (Type I, Type II) or acombination thereof, BCG Sweden (1927), BCG Birkhaug, BCG Prague, BCGGlaxo (1954), BCG Merieux (1989), BCG Danish, BCG Frappier, BCGConnaught (1948), BCG Mexico, BCG Tice (1934), BCG China or BCG Phipps.

In a preferred embodiment, the Mycobacterium for use in the vaccinecompositions of the present invention is a Tokyo strain of BCG. In oneembodiment, the Mycobacterium for use in the vaccine compositions of thepresent invention is BCG Tokyo 172 (ATCC 35737; TMC1019). The BCG Tokyostrain for use in the vaccine compositions provided herein can be theType I or Type II subpopulations or a combination or mixture thereof. Itshould be noted that the two subpopulations differ in their colonymorphologies with Type I being smooth and Type II being rough. Further,smooth colonies have a characteristic 22-bp deletion in Rv3405c of theregion of difference (RD) 16 (type I), while rough colonies are completein this region (type II). Additionally, the subpopulation types alsodiffer in their lipid phenotypes, with phenolic glycolipid (PGL) andphthiocerol dimycocerosate (PDIM) being found only in type I. Given thatPGL has been shown to suppress the host recognition of total lipids viaToll-like receptor 2, it may be antigenic and involved in hostresponses, acting as a cell wall component.

In one embodiment, the strain of BCG present in a vaccine composition asprovided herein is a genetically engineered strain of BCG. The strain ofBCG can be genetically engineered to improve or enhance immune systemfunction such as, for example, T cell-mediated immunity. The strain ofBCG can be genetically engineered to be equipped with themembrane-perforating listeriolysin (Hly) of Listeria monocytogenes asshown in Grode et al. Increased vaccine efficacy against tuberculosis ofrecombinant Mycobacterium bovis bacille Calmette-Guérin mutants thatsecrete listeriolysin, J. of Clinical Investigation, Vol. 115: 9, Sep.2005, which is herein incorporated by reference in its entirety.

In one embodiment, the Mycobacterium is non-pathogenic. Thenon-pathogenic Mycobacterium can be heat-killed. The non-pathogenicMycobacterium can be selected from M. vaccae, M. obuense, M.parafortuitum, M. aurum, M. indicus pranii, M. phlei and combinationsthereof. In one embodiment, the non-pathogenic Mycobacterium is a roughvariant. In one embodiment, the non-pathogenic Mycobacterium is a smoothvariant. In one embodiment, the methods provided herein compriseadministering a composition comprising M. vaccae. Further to thisembodiment, the M. vaccae can be heat-inactivated. Further still to thisembodiment, the M. vaccae can be strain NCTC11659. Even further still tothis embodiment, the composition comprising M. vaccae is SRL172. SRL172is a suspension of heat-killed whole cell Mycobacterium vaccae. In oneembodiment, the methods provided herein comprise administering acomposition comprising M. obuense. Further to this embodiment, the M.obuense can be heat-inactivated. Further still to this embodiment, theM. obuense can be strain NCTC13365. Even further still to thisembodiment, the composition comprising M. obuense is IMM-101. IMM-101 isa suspension of heat-killed whole cell Mycobacterium obuense.

In one embodiment, the compositions provided herein can be used asvaccines and can accordingly be formulated as pharmaceuticalcompositions.

In one embodiment, administration of any vaccine composition providedherein (e.g., a vaccine composition comprising an isolated Mycobacteriumor antigenic fragment thereof) reduces, eliminates or alleviates one ormore symptoms of FM. The one or more symptoms can be selected fromchronic muscle pain, muscle spasms, or tightness, moderate or severefatigue and decreased energy, insomnia or feeling of exhaustion,stiffness upon waking or after staying in one position for too long,difficulty remembering, concentrating, and performing simple mentaltasks (“fibro fog”), abdominal pain, bloating, nausea, and constipationalternating with diarrhea (irritable bowel syndrome), tension ormigraine headaches, jaw and facial tenderness, sensitivity to one ormore of odors, noise, bright lights, medications, certain foods, andcold, feeling anxious or depressed, numbness or tingling in the face,arms, hands, legs, or feet, increase in urinary urgency or frequency(irritable bladder), reduced tolerance for exercise and muscle painafter exercise. a feeling of swelling (without actual swelling) in thehands and feet or a combination thereof. The reduction, elimination oralleviation can be as compared to a control. The control can be theindividual prior to administration of the vaccine composition or aseparate individual suffering from FM. In one embodiment, the reduction,elimination or alleviation of the one or more symptoms can be determinedby evaluating the individual according to the Fibro-fatigue scale.

In another embodiment, administration of any vaccine compositionprovided herein (e.g., the vaccine composition comprising an isolatedMycobacterium or antigenic fragment thereof) increases immune systemfunctioning. The increase in immune system functioning can be evidencedby the production or elevation thereof of T_(H)1 cytokines, upregulationof granzyme B or both. The T_(H)1 cytokines that are elevated orproduced in response to administration of the compositions providedherein can include IFN-γ, IL-2, or TNF-β or a combination thereof. Theincrease immune system function can be evidenced by an elevation orincrease in the production of one or more cytokines provided herein suchas, for example, the cytokines listed in Table 1. Assessment of thealteration in immune function can be ascertained using the methodsand/or kits provided herein and/or as described in US20150301062.

In one embodiment, administration of any vaccine composition providedherein (e.g., a vaccine composition comprising an isolated Mycobacteriumor antigenic fragment thereof) to an individual suffering from orsuspected of suffering from CFS reduces, eliminates or alleviates one ormore symptoms of CFS. The one or more symptoms can be selected from asubstantial reduction or impairment in the ability to engage inpre-illness levels of occupational, educational, social, or personalactivities, post-exertional malaise, unrefreshing sleep, cognitiveimpairment, orthostatic intolerance, pain, gastrointestinal andgenitourinary problems, sore or scratchy throat, painful or tenderaxillary/cervical lymph nodes, sensitivity to external stimuli, immunesystem problems, hormonal imbalances or any combination thereof. Thereduction, elimination or alleviation can be as compared to a control.The control can be the individual prior to administration of the vaccinecomposition or a separate individual suffering from CFS. In oneembodiment, the reduction, elimination or alleviation of the one or moresymptoms can be determined by evaluating the individual according to theFibro-fatigue scale.

In another embodiment, administration of any vaccine compositionprovided herein (e.g., the vaccine composition comprising an isolatedMycobacterium or antigenic fragment thereof) to an individual sufferingfrom or suspected of suffering from CFS increases immune systemfunctioning. The increase in immune system functioning can be evidencedby the production or elevation thereof of T_(H)1 cytokines, upregulationof granzyme B or both. The T_(H)1 cytokines that are elevated orproduced in response to administration of the compositions providedherein can include IFN-γ, IL-2, or TNF-β or a combination thereof. Theincrease immune system function can be evidenced by an elevation orincrease in the production of one or more cytokines provided herein suchas, for example, the cytokines listed in Table 1. Assessment of thealteration in immune function can be ascertained using the methodsand/or kits provided herein and/or as described in US20150301062.

Administration of Compositions

In certain embodiments, the compositions described herein (e.g., theimmunogenic vaccine compositions) comprise, or are administered incombination with, an adjuvant. The adjuvant for administration incombination with a composition described herein may be administeredbefore, concomitantly with, or after administration of said composition.In some embodiments, the term “adjuvant” can refer to a compound thatwhen administered in conjunction with or as part of a compositiondescribed herein augments, enhances and/or boosts the immune response toan antigen present in the composition (e.g., the isolated Mycobacteriumor antigenic fragment derived therefrom) but when the compound isadministered alone does not generate an immune response to the antigenpresent in the composition (e.g., the isolated Mycobacterium orantigenic fragment derived therefrom). In some embodiments, the adjuvantgenerates an immune response to the antigen present in the composition(e.g., an isolated Mycobacterium or antigenic fragment derivedtherefrom) and does not produce an allergy or other adverse reaction.Adjuvants can enhance an immune response by several mechanismsincluding, e.g., lymphocyte recruitment, stimulation of B and/or Tcells, and stimulation of macrophages. When a vaccine or immunogeniccomposition of the invention comprises adjuvants or is administeredtogether with one or more adjuvants, the adjuvants that can be usedinclude, but are not limited to, mineral salt adjuvants or mineral saltgel adjuvants, particulate adjuvants, microparticulate adjuvants,mucosal adjuvants, and immunostimulatory adjuvants. Examples ofadjuvants for use in the methods and compositions provided herein caninclude, but are not limited to, cytokines (e.g., IL-12), heat-shockproteins, aluminum salts (alum) (such as aluminum hydroxide, aluminumphosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A(MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), ASO4(GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.),imidazopyridine compounds (see International Application No.PCT/US2007/064857, published as International Publication No.WO2007/109812), imidazoquinoxaline compounds (see InternationalApplication No. PCT/US2007/064858, published as InternationalPublication No. WO2007/109813) and saponins, such as QS21 (see Kensil etal., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell &Newman, Plenum Press, N Y, 1995); U.S. Pat. No. 5,057,540). In someembodiments, the adjuvant is Freund's adjuvant (complete or incomplete).Other adjuvants are oil in water emulsions (such as squalene or peanutoil), optionally in combination with immune stimulants, such asmonophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91(1997)).

The compositions provided herein can comprise an antigen (e.g., anisolated Mycobacterium or antigenic fragment derived therefrom) aloneor, preferably, together with a pharmaceutically acceptable carrier.Suspensions or dispersions of an antigen (e.g., an isolatedMycobacterium or antigenic fragments derived therefrom), especiallyisotonic aqueous suspensions or dispersions, can be used. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, e.g., preservatives, stabilizers, wetting agents and/oremulsifiers, solubilizers, salts for regulating osmotic pressure and/orbuffers and are prepared in a manner known per se, for example by meansof conventional dispersing and suspending processes. The saiddispersions or suspensions may comprise viscosity-regulating agents. Thesuspensions or dispersions are kept at temperatures around 2-4° C., orpreferentially for longer storage may be frozen and then thawed shortlybefore use. For injection, the vaccine or immunogenic preparations maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents.

In certain embodiments, the compositions described herein additionallycomprise a preservative, e.g., the mercury derivative thimerosal. In aspecific embodiment, the pharmaceutical compositions described hereincomprises 0.001% to 0.01% thimerosal. In other embodiments, thepharmaceutical compositions described herein do not comprise apreservative.

The compositions of the invention may be administered to mammals (e.g.,rodents, humans) in any suitable formulation. For example, isolatedMycobacterium or antigenic fragments thereof may be formulated inpharmaceutically acceptable carriers or diluents such as physiologicalsaline or a buffered salt solution. Suitable carriers and diluents asprovided herein can be selected on the basis of mode and route ofadministration and standard pharmaceutical practice.

The compositions of the invention may be administered to mammals by anyconventional technique. Typically, such administration will be oral,sublingual, nasal, pulmonary or parenteral (e.g., intravenous,subcutaneous, intravesicular, intramuscular, intraperitoneal,intradermal, subdermal, or intrathecal introduction). The compositionsmay also be administered directly to a target site by, for example,surgical delivery to an internal or external target site, or by catheterto a site accessible by a blood vessel. The compositions may beadministered in a single bolus, multiple injections, or by continuousinfusion (e.g., intravenously, by peritoneal dialysis, pump infusion).For parenteral administration, the compositions are preferablyformulated in a sterilized pyrogen-free form.

Dosing

The compositions (e.g., vaccine compositions) described above arepreferably administered to a mammal (e.g., a human) in an effectiveamount, that is, an amount capable of producing a desirable result in atreated individual (e.g., activating or boosting the immune response).Such a therapeutically effective amount can be determined as describedbelow.

Toxicity and therapeutic efficacy of the compositions utilized inmethods of the invention can be determined by standard pharmaceuticalprocedures, using either cells in culture or experimental animals todetermine the LD₅₀ (the dose lethal to 50% of the population). The doseratio between toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio LD₅₀/ED₅₀. Those compositions thatexhibit large therapeutic indices are preferred. While those thatexhibit toxic side effects may be used, care should be taken to design adelivery system that minimizes the potential damage of such sideeffects. The dosage of preferred compositions lies preferably within arange that includes an ED₅₀ with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

As is well known in the medical and veterinary arts, dosage for any onesubject depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, timeand route of administration, general health, and other drugs beingadministered concurrently.

In certain embodiments, a particular dosage of a vaccine compositionprovided herein (e.g., vaccine comprising a strain of BCG) isadministered to a subject. In certain embodiments of the invention,there is provided a vaccine composition comprising a live-attenuated orheat-killed bacteria (e.g., strain of BCG) for use in the presentinvention, which typically may be from 10³ to 10¹¹ cells or colonyforming units (CFUs), from 10⁴ to 10¹⁰ cells or CFUs, from 10⁶ to 10¹⁰cells or CFUs, or 10⁶ to 10⁹ cells or CFUs per unit dose. The effectiveamount of live-attenuated or heat-killed Mycobacterium for use in themethods or compositions provided herein can be from 10³ to 10¹¹ cells orCFUs, from 10⁴ to 10¹⁰ cells or CFUs, from 10⁶ to 10¹⁰ cells or CFUs,and from 10⁶ to 10⁹ cells or CFUs per unit dose. The unit dose can be 5ul, 10 ul, 20 ul, 30 ul, 40 ul, 50 ul, 60 ul, 70 ul, 80 ul, 90 ul, 100ul, 125 ul, 150 ul, 175 ul, 200 ul, 250 ul, 300 ul, 350 ul, 400 ul, 450ul, 500 ul, 600 ul, 650 ul, 700 ul, 750 ul, 800 ul, 850 ul, 900 ul, 950ul, 1000 ul or 1500 ul. In one embodiment, the vaccine comprises atherapeutically effective amount of live-attenuated or heat-killedMycobacterium (e.g., strain of BCG such as Tokyo Strain) is from 1.8×10⁶to 3.9×10⁶ colony forming units per unit dose, wherein the unit dose is0.1 ml. Alternatively, the dose of a vaccine composition provided hereincan be from 0.01 mg to 1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1 mg, 1 mg to1.5 mg or 1.5 mg to 2.0 mg. In one embodiment, the dose is 1 mg. In oneembodiment, the dose is 0.5 mg. In one embodiment, the dose is 0.1 mg.The organisms or antigenic fragments derived therefrom can be presentedas either a suspension or dry preparation. Further to the aboveembodiments, the route of administration can be intradermal (ID)administration.

The composition may advantageously further comprise vitamin B₁₂ and/orfolacin. It has been found that a subgroup of patients suffering fromfibromyalgia or chronic fatigue syndrome, may also have levels ofvitamin B₁₂ in their cerebrospinal fluid that are lower than normal, andlevels of homocysteine that are higher than normal.

The composition according to the invention may also comprise, such aspharmaceutically acceptable additives, e.g. solvents, adjuvants,carriers and/or preservatives as provided herein.

The methods of treatment for FM, CFS and/or related conditions asprovided herein can be conducted as a series of administrations withincreasing doses during a specific period. In one example, the vaccinecomposition can be administered in 8-10 increasing doses during 4-12weeks, preferably 8-10 weeks. The reason for the increasing doses can bethat during the first week or weeks the patient may suffer from sideeffects, and it is therefore advantageously to start with a low dose.The side effects may diminish after some time. In one embodiment, thevaccine composition can be administered in 2 doses spaced 4 weeks apart.

In order to obtain the desired effect for a prolonged period of time thevaccine preparation (e.g., vaccine comprising BCG) may be administeredat several occasions. For example, a first series of administrations maybe followed by repeated administrations given at specified intervals.The specified intervals can be approximately once a week for 5-15 weeks,preferably for 10 weeks.

To prevent recurrence, the repeated administrations may then be followedby a maintenance treatment with administrations at specified intervals.The specified intervals can be approximately once a month. The specifiedintervals may be continued for several years, such as 1-10 years, orapproximately 5 years. In one embodiment, the maintenance treatmententails one injection of the vaccine composition per year for aspecified interval of 4 years.

The doses in the repeated administrations of the maintenance treatmentcan be constant. In some cases, the doses in the maintenance treatmentcan be the dose used in the last administration in the first series.

These repeated administrations can result in an unspecific or specificactivation of the immune system over a long period of time.

The administrations can be made in any way known in the art, such as,for example, injections.

Additional agents or substances can be administered simultaneously or inparallel with the vaccine compositions of the present invention. Theadditional agents or substance can be, for example, vitamin B12 and/orfolacin.

Assessing Treatment Efficacy

In another aspect, the invention provides methods for evaluating theefficacy of treatment in an individual diagnosed with FM or CFS. In oneembodiment, the method for evaluating treatment efficacy in anindividual diagnosed with FM entails subjecting the individual to theFibroFatigue scale following treatment or at various points duringtreatment. In one embodiment, the method for evaluating treatmentefficacy in an individual diagnosed with CFS entails evaluating theindividual diagnosed with CFS, following treatment or at various pointsduring treatment, for improvements in the set of symptoms used to makethe CFS diagnosis such as, for example, evaluating any improvement inactivities of daily living and/or improvements in physical and mentalfunctioning. Alone or in combination with the previous two embodiments,the method for evaluating the efficacy of treatment in an individualdiagnosed with FM or CFS involves determining or detecting as a baselinethe level of one or more cytokines expressed in the individual diagnosedwith or suspecting of having FM or CFS prior to treatment. Followingtreatment (e.g., vaccination), subsequent measurements of one or morecytokine levels are carried out to determine the levels or patterns ofexpression of the one or more cytokines. The altered levels and/orpatterns of expression of one or more of the cytokines measured in theindividual afflicted with FM or CFS or at risk for developing FM or CFSand undergoing treatment are compared to the levels or patterns ofexpression of cytokines in a control. In one embodiment, the control isthe levels and/or patterns of expression of the one or more cytokines inthe individual before treatment. In another embodiment, the control isthe levels and/or expression levels of the one or more cytokines from ahealthy patient, or cytokine levels reported for a patient withoutfibromyalgia or CFS (for example, levels reported in a database).

In one embodiment, the methods for assessing treatment efficacy involvedetermining or assaying the levels of at least one, at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, or at least ten or more cytokines in theplasma of blood samples obtained from individuals suspected of beingafflicted with FM or CFS or at risk for FM or CFS after treatment withthe compositions (e.g., the compositions comprising isolatedMycobacterium or antigenic fragments thereof) and comparing the levelsof the assayed cytokines to a control. The control can be any control asprovided herein. In a further embodiment, the method involvesdetermining or assaying the levels of at least one, at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, or at least ten or more cytokines in theperipheral blood mononuclear cells (PBMCs) that have been separated fromthe plasma of blood samples obtained from the individuals aftertreatment and comparing the levels of the assayed cytokines to acontrol. The control can be any control as provided herein. These levelsare then analyzed to determine if the levels are altered due to thetreatment. For example, the levels in the individual's sample duringand/or after treatment, in one embodiment, are compared to levels in acontrol sample, for example, a sample known to not have FM or CFS. Inanother embodiment, control levels are known, for example, from adatabase. In one embodiment, a change in expression in a majority of thecytokines tested toward the levels in the control isdeterminative/indicative of the treatment for FM or CFS beingefficacious. In another embodiment, a change in expression of at leastabout 33% or at least about 67% of the cytokines tested isdeterminative/indicative of an effective treatment for FM or CFS. In afurther embodiment, a treatment with a composition as provided herein isdeemed to be efficacious for treating fibromyalgia or CFS if at leastabout 75%, or at least about 75% or more of the cytokines tested havealtered expression due to the treatment. In even a further embodiment, atreatment with a composition as provided herein is deemed to beefficacious for treating fibromyalgia or CFS if the expression level ofevery cytokine tested, or about every cytokine tested in the patient isaltered. The altered expression of one or more cytokines during orfollowing treatment can be modifying the level or expression of the oneor more cytokines to be substantially the same expression level of theone or more cytokines in a control (e.g., the levels in a healthypatient who does not have FM or CFS). As used herein, the term“substantially the same expression level” can be about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 99% or about 99% of the expression level of a particularcytokine in a control as provided herein.

The present invention is not limited by any particular combination ofcytokines. For example, the cytokines to whose expression can beevaluated in order to determine treatment efficacy can be selected fromIL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15,IL-17, IL-21, IFN-γ, IFN-α, TNF-α, IP-10, MCP-1, MIG, MIP-1α, MIP-1β,GM-CSF, Eotaxin, RANTES, etc. or a combination thereof. In anotheraspect, the invention further includes determining the levels of one ormore of IL-IRA, IL2R, IL-7, IL-12 (p40/p70), IL-13, IL-15, IL-17, IFN-α,IP-10, MIG, VEGF, G-CSF, EGF, granzyme B, FGF-basic and HGF or acombination thereof. In yet another aspect, the invention also includesdetermining the levels of IL-9 and PDGF-BB or a combination thereof. Thecytokine may be inflammatory or anti-inflammatory. In one embodiment,the cytokine to be assayed may be a full length polypeptide, protein, aglycoprotein or a fragment thereof. Other proteins that can be assayedinclude hormones, heat-shock proteins, antibodies such as but notlimited to anti-nuclear antibody (ANA), thyroid antibodies,anti-extractable nuclear antibodies (ENA), IgG subclasses, anti-nuclearfactors (FAN), rheumatoid factor (RF), receptor proteins and ligands,etc. In other embodiment, the level of cytokine assayed maybe a mRNA,miRNA, or DNA. In another example, the expression levels of cytokinesincluded in commercial cytokine panels (or cytokine subsets thereof) canbe evaluated by the methods provided herein. Various combinations ofcytokines for use in the present invention are provided in the Table 1as provided herein. Subsets of these combinations may also be used inthe methods provided herein. It should be understood that thesecombinations are representative, and should not be construed as limitingthe invention.

Measurement/Detection of Cytokine Levels

In one embodiment, cytokine levels in methods entailing diagnosingand/or assessing treatment efficacy as provided herein are tested on theprotein level. In another embodiment, cytokine levels in methodsentailing diagnosing and/or assessing treatment efficacy as providedherein are determined at the mRNA level. In yet another embodiment, bothmRNA and protein levels for the cytokines are examined in the methodsprovided herein. Methods for assaying cytokines at the protein or mRNAlevels are well known in the art and can be employed in the methodsprovided herein.

Measuring cytokine levels in methods entailing diagnosing and/orassessing treatment efficacy as provided herein can be from blood or aplasma sample that may be stimulated or un-stimulated. That is, cellproliferation may be induced prior to assaying the cytokine levels. Inone embodiment, the PBMCs are un-stimulated. In another embodiment, thePBMCs are stimulated to cause proliferation of the cells prior toassaying for cytokines. Methods for stimulating PBMCs are known in theart, and include, but are not limited to, the addition of mitogens tothe cells. Non-limiting examples of mitogens include lipopolysaccharide(LPS), phytohemagglutinin (PHA), or phorbol ester, such as phorbolmyristate acetate (PMA) with or without ionomycin, pokeweed mitogen(PWM), concavalin A (Con-A), or combinations thereof.

In one embodiment, cytokine expression is measured at the mRNA level,for example, by quantitative RT-PCR (also known as real time RT-PCR).mRNA expression levels can also be measured by Northern blot assay,array hybridization, sequencing, etc. For example, multiplexquantitative RT-PCR, in one embodiment, is carried out to determine themRNA expression levels of a cytokine panel. Cytokine RT-PCR kits arecommercially available, for example, from Roche.

In another embodiment, secreted cytokine levels are determined (i.e., atthe protein level). In one embodiment, secreted cytokine levels aredetermined by using an antibody array, for example, the TranSignal HumanCytokine Antibody Array 3.0, available from Panomics. The Panomics arrayincludes antibodies directed to the following cytokines: Apol/Fas,Leptin, Rantes, ICAM-1, IL-2, IL-7, CTLA, MIP-1α, MIP-1β, TGFβ, VCAM-1,IL-3, IL-8, IL-4, IL-10, IL-5, IL-12, IL-6, IL-15, IL-6R, IL-17, IL-1Rα,IL-1β, IL-1α, VEGF, IFNγ, TNFγ, TNFRI, TNFRII, MIP-5, MIP-4, MMP3,Eotaxin, GM-CSF, EGF, IP-10. In this embodiment, not all cytokines inthe array need be probed for. For example, the expression levels of asubset of five cytokines, or five or more cytokines, or six cytokines,or six or more cytokines, or seven cytokines, or seven or morecytokines, or ten cytokines, or ten or more cytokines, or twelvecytokines, or twelve or more cytokines may be determined when carryingout the methods of the invention.

Secreted cytokine levels, in one embodiment, are determined with amultiplex immunoassay built on magnetic beads. For example, in oneembodiment, the Bio-Plex Pro magnetic Cytokine Assay is used (Bio-Rad).In this embodiment, the Assay is commercially available as a ready touse kit, for example, for the detection of eight cytokines, seventeencytokines, 21 cytokines or 27 cytokines. The full number or a subset ofthe cytokines may be detected in the methods of the invention.Alternatively, expression levels of cytokines can be tested in a sampleby doing multiple assays on the sample, for example, in “singleplex”format. In one embodiment, the Bio-Rad singleplex cytokine assays areused.

Another antibody based bead assay is available from Invitrogen, and isalso amenable to be used in the methods of the present invention.Specifically, the Human Cytokine Thirty-Plex antibody bead kit may beemployed to detect the levels of a panel of cytokines in an individual.Although the assay can detect the levels of thirty cytokines, not allthirty need to be detected in order to carry out the methods providedherein. For example, as provided above, five, six, seven, eight, nine,ten, eleven or twelve cytokines can be assayed for their expressionlevels. The Invitrogen kit comprises analyte specific components for themeasurement of human IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ,GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, G-CSF,EGF, FGF-basic, and HGF. These reagents, in one embodiment, are used inthe Luminex® 100™ or 200™ System.

Methods for assaying cytokines at the protein or mRNA levels are wellknown in the art. Besides the assays provided above, other non-limitingexamples of methods for assaying cytokines at the protein level includeenzyme-linked immunoassay (ELISA), Tetramer assay, ELISPOT assay,Fluorospot assay, etc. The cytokines concentration in the plasma,culture supernatant, or cell lysate derived from PBMC can be measured,for example, by multiplex immunoassay based on Luminex xMAP bead arraytechnology, or Bio-Plex 200 fluorescence bead reader (BioRadLaboratories, Hercules, Calif.). In one embodiment, the level of one ormore cytokine mRNA can be detected (measured) by real time PCR, RT-PCR,Northern blot assay, array hybridization, sequencing, etc. The alteredlevel(s) of the cytokines measured in the affected individual comparedto the level from control group is predictive/indicative of FM in theindividual. The cytokine levels in an individual with FM, for example,cytokine levels in a FM patient's blood, in one embodiment, are higherthan the cytokine levels of a healthy patient, for each cytokine tested.In another embodiment, the cytokine levels in a FM patient's blood arelower than the cytokine levels of a healthy patient, for each cytokinetested. In yet another embodiment, the cytokine levels measured in apatient with FM may be higher or lower, depending on the panel ofcytokines measured in the individual.

The level of cytokines can be determined using an algorithm and the rawdata obtained by measuring the levels of cytokines which have beenstored in a computer system, or any other medium that is linked to acomputer or machine. In one aspect, the method further includesevaluation of the individual's (i.e., individuals diagnosed with FM orCFS) clinical and physical symptoms as described herein in conjunctionwith determining the levels of one or more cytokines. For example, in anindividual diagnosed with FM, the method can include evaluation ofphysical and mental functioning as well as tender points in theindividual. The physical and mental functioning as well as the painthreshold can be calculated and assigned a score on a subjective basis.In another example, in an individual diagnosed with CFS, the method caninvolve evaluation of the set of symptoms used to make the originaldiagnosis such as, for example, evaluating the individual's dailyactivity and/or physical and mental functioning. The level of dailyactivity as well as the physical and mental functioning can becalculated and assigned a score on a subjective basis. The scoresderived from the assessment of the clinical and physical symptoms may beincluded in the statistical analysis for the cytokines. In a furtherembodiment, the method includes determining the levels of variousfactors or markers, such as but not limited to Rheumatoid Factor (RF),or a specific marker of inflammation such as the erythrocytesedimentation rate (ESR).

As it relates to the diagnostic and/or assessment of treatment efficacymethods provided herein, cytokine expression can be “altered” or“differentially expressed”, in an individual, in one embodiment, ifexpression of the cytokine in the individual's sample is at least about1.5 times higher or lower than the expression of the same cytokine at acontrol level. In another embodiment, cytokine expression is “altered”if cytokine expression in the individual's sample is at least about 2times higher or lower than the expression of the same cytokine at acontrol or baseline level (i.e., levels reported for a healthy patient).In another embodiment, cytokine expression is “altered” if cytokineexpression in the individual is at least about 2.5 times higher or lower(or at least about 2.5 times or more higher or lower) than the controlexpression level of the same cytokine. In yet another embodiment,cytokine expression is “altered” if cytokine expression in theindividual is at least about 3 times higher or lower (or at least about3 times or more higher or lower) than the control expression level ofthe same cytokine. In another embodiment, cytokine expression is“altered” if cytokine expression in the individual is at least about 5times higher or lower (or at least about 5 times or more higher orlower) than the control expression level of the same cytokine. In evenanother embodiment, cytokine expression is “altered” if cytokineexpression in the individual is at least about 10 times higher or lowerthan the control expression level of the same cytokine in a controlsample. In yet another embodiment, cytokine expression is altered ifcytokine expression in the individual is at least about 10 times ormore, higher or lower, than the control expression level of the samecytokine. As provided above, control expression level may be determinedfrom values in a database, from a non-disease sample (e.g., FM or CFS)or individual.

Altered expression of the cytokine may be the same or different for eachindividual cytokine that is differentially expressed. For example, theexpression of one cytokine (mRNA or protein) may be 2× lower, or about2× lower, than the expression of the same cytokine in a control sample,while the expression of a second cytokine may be 1.5× lower, or about1.5× lower, than the expression of the same cytokine in a controlsample. As discussed above, altered expression includes both higher andlower expression of the cytokine, compared to a control level.

EXAMPLES Example 1: A Placebo-Controlled Study to Evaluate the Safetyand Efficacy of Mycobacterium Vaccines in Treating Fibromyalgia (FM)

The primary objective of this study will be to evaluate the efficacy oftwo heat-killed whole cell Mycobacterium compositions for boosting theimmune system of patients suffering from fibromyalgia (FM). Secondaryobjectives will be to evaluate the safety and tolerability of both ofthe Mycobacterium compositions as well as investigate local injectionsite reactions.

Study Patients and Dosing Schedule

The study will be in the form of a prospective, randomized,double-blind, placebo-controlled, parallel-group study using differentMycobacterium comprising compositions. Male and female patients (over 18years) who have a positive FM/a® score as assessed using the FM/a®fibromyalgia blood test are included in this trial. The main exclusioncriteria will include pregnant and lactating woman, patients sufferingfrom other inflammatory rheumatological diseases (such as rheumatoidarthritis or collagenoses), severe neuropathies, clinically manifestendocrinopathies, bone diseases, severe cardial, renal or hepaticimpairment and acute or chronic infections.

75 Patients will be randomly assigned to one of three study cohorts:placebo (25 patients; cohort 1), IMM-101 (M. obuense composition; 25patients; cohort 2) or SRL172 (M. vaccae composition; 25 patients;cohort 3). The duration of treatment will be four weeks over which eachpatient will receive (3) intradermal (i.d.) injections. Each dose willbe administered in each patient from one of the three cohorts into theskin overlying the deltoid muscle with the arm alternated for each dose.Prior to commencement of the study, each patient in each cohort willreceive a placebo injection of borate saline solution (day −3) toprovide an intra-patient placebo control and to allow the patient topractice completion of the diary and assess whether patients are capableof measuring their own in injection site reactions accurately. Patientswho are willing and able to proceed with the study will be injected witha single dose level of placebo (borate saline-solution, cohort 1);IMM-101 (cohort 2) or SRL172 (cohort 3) on (3) subsequent occasions.Doses of placebo, IMM-101 or SRL172 will be administered over a 4-weekperiod on days 0, 14 and 28 (with up to 2 days variation in the dosinginterval). For cohort 1, a standard volume of 0.1 ml of boratesaline-solution will be injected. For cohort 2, a standard volume of 0.1ml of a suspension containing M. obuense at the concentration of 10mg/ml will be injected. For cohort 3, a standard volume of 0.1 ml of asuspension containing 10⁹ bacilli of M. vaccae will be injected.

Before, on each dosing day and at the end of the treatment phase (i.e.,at screening and at days 3, 14, 28 and 42), routine safety assessmentswill be performed using physical examinations, urinalysis,electrocardiograms and hematological and biochemical blood tests.Further, at days 0, 3, 14, 28 and 42, local tolerability will beassessed using standardized techniques (measurements at injection site)by a study physician or research nurse and the intensity of eachinjection site reaction will be scored with reference to a VaccineToxicology Rating Scale (available at Annals of Oncology online).Additionally, at screening and at days 3, 14, 28 and 42, a painassessment and blood testing for cytokine levels will be performed inorder to assess physical signs and symptoms of FM as well as thebiochemical effects of the vaccination protocol, respectively.

In order to document daily the intensity of pain, adverse events andconcomitant medications, patients will use a standardized diary and willrecord daily the parameters mentioned. In addition, changes infunctional symptoms will be documented at start of treatment, as well ason day 3, day 14, day 28 and at the end of treatment (day 42). Adverseevents will be assessed during the active treatment period.

To evaluate pain, the pain score, a visual analogue scale and clinicalexamination of tender points will be used. The pain score ranges from 0to 120, measuring the pain intensity in 24 body regions applied to thefollowing rating scale: 0=no pain, 1=mild pain, 2=moderate pain,3=moderately severe pain, 4=severe pain, 5=most ever pain. Theassessment of each body region will be done by the patients themselves;the total score will be calculated as the sum of the regional scores.

The visual analogue scale is in the form of 100-mm-line orientedhorizontally with one end=0, indicating “no pain” and the other end=100,indicating “worst pain”. The patients are asked to place a markcorresponding to their perception of their present pain intensity.

In addition to the documented effects during the active treatment phase,a follow-up of the patients will be performed for six months in order toevaluate the duration of the clinical response (as defined by a 35% orhigher reduction in individual pain score/baseline versus end oftreatment).

Cytokine Assay

In order to measure cytokine levels (i.e., IL-2 and IFN-gamma), atscreening and at days 3, 14, 28 and 42, twenty-30 mL of blood will bedrawn from each patient from each cohort in 7-mL tubes containing 0.081mL of 15% K₃-EDTA solution (BD Vacutainer®, BD, Franklin Lakes, N.J.) byvenipuncture. Blood will be transferred to 50-mL tubes and diluted 1:1in GIBCO® Hank's balanced salt solution (HBSS, Invitrogen, Carlsbad,Calif.). The resulting solution will be layered on top of 15 mL Ficoll(Histopaque®-1077, Sigma-Aldrich, St. Louis, Mo.) in 50-mL tubes andcentrifuged at 1800 rpm for 20 minutes. The top layer will aspirated anddiscarded and the interphase containing PMBC will be harvested andtransferred into a new 50-mL tube. The tube will be filled with HBSS andthe contents mixed by gentle rocking. Cells will be collected bycentrifugation at 2000 rpm for 10 minutes and resuspended in 10 mLcomplete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1%penicillin-streptomycin mixture and 1% L-glutamine solution (Invitrogen,Carlsbad, Calif.). Cell viability and count will be determined usingTrypan blue exclusion assay.

Cytokine and chemokine concentrations in plasma will be measured bymultiplex immunoassay based on Luminex xMAP™ bead array technology andBio-Plex 200 fluorescence bead reader (BioRad Laboratories, Hercules,Calif.). Three panels of antibody-conjugated beads for measuring humaninflammatory cytokines (GM-CSF, IL-1β, IL-6, IL-8, TNF-α), Th1/Th2cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-10) and chemokines (MIP-1α,MIP-1β, MCP-1, Eotaxin, RANTES) (BioSource, Camarillo, Calif.) will beused in the assay according to the manufacturer instructions. Otherpanels that can be included are IL-1RA, IL-2R, IL-7, IL-12 (p40/p70),IL-13, IL-15, IL-17, IFN-, IP-10, MIG, VEGF, EGF, FGF-basic, and HGF(InVitrogen) or IL-IRA, IL-7, IL-9, IL-12, IL13, IL-15, IL-17,FGF-basic, G-CSF, IP-10, VEGF, PDGF-BB (BioRad)

Concentration values will be transferred to Microsoft Office Excel 2003software (Microsoft Corporation, Redmond, Wash.) and means as well asstandard deviations will be calculated for each cytokine concentration.Cytokine concentrations in patients from cohorts 2 and 3 will becompared to those of cohort 1 by using Student's t-test. The confidencelevel will be set at 5%. In addition, analysis of a potentialcorrelation between cytokine induction (i.e., IL-2 and/or IFN-gamma) andmodification of physical/function FM signs & symptoms will be performedusing software and statistical analyses.

Example 2: A Placebo-Controlled Study to Evaluate the Safety andEfficacy of a BCG Vaccine in Treating Fibromyalgia (FM)

The primary objective of this study will be to evaluate the efficacy ofa BCG vaccine for boosting the immune system of patients suffering fromfibromyalgia (FM). Secondary objectives will be to evaluate the safetyand tolerability of both of the BCG vaccine as well as investigate localinjection site reactions.

Study Patients and Dosing Schedule

The study will be in the form of a prospective, randomized,double-blind, placebo-controlled, parallel-group study using a BCGvaccine. Male and female patients (over 18 years) who have a positiveFM/a® score as assessed using the FM/a® fibromyalgia blood test areincluded in this trial. The main exclusion criteria will includepregnant and lactating woman, patients suffering from other inflammatoryrheumatological diseases (such as rheumatoid arthritis or collagenoses),severe neuropathies, clinically manifest endocrinopathies, bonediseases, severe cardial, renal or hepatic impairment and acute orchronic infections.

50 Patients will be randomly assigned to one of two study cohorts:placebo (25 patients; cohort 1) or BCG vaccine (25 patients; cohort 2).The BCG used will be Tokyo BCG (JBL, Tokyo, Japan). The duration oftreatment will be four weeks over which each patient will receive (2)intradermal (ID) injections. Each dose will be administered in eachpatient from one of the two cohorts into the skin overlying the deltoidmuscle with the arm alternated for each dose. Prior to commencement ofthe study, each patient in each cohort will receive a placebo injectionof borate saline solution (day −3) to provide an intra-patient placebocontrol and to allow the patient to practice completion of the diary andassess whether patients are capable of measuring their own in injectionsite reactions accurately. Patients who are willing and able to proceedwith the study will be injected with a single dose level of placebo(borate saline-solution, cohort 1) or BCG (cohort 2) on (2) subsequentoccasions. Doses of placebo or BCG will be administered over a 4-weekperiod on days 0 and 28 (with up to 2 days variation in the dosinginterval). For cohort 1, a standard volume of 0.1 ml of boratesaline-solution will be injected. For cohort 2, a standard volume of 0.1ml of a suspension containing BCG at the concentration of 1.8 to 3.9×10⁶cfu will be injected. All injections will be administered using aBiojector® B2000 device for intradermal (ID) administration.

Before, on each dosing day and at the end of the treatment phase (i.e.,at screening and at days 3, 14, 28 and 42), routine safety assessmentswill be performed using physical examinations, urinalysis,electrocardiograms and hematological and biochemical blood tests.Further, at days 0, 3, 14, 28 and 42, local tolerability will beassessed using standardized techniques (measurements at injection site)by a study physician or research nurse and the intensity of eachinjection site reaction will be scored with reference to a VaccineToxicology Rating Scale (available at Annals of Oncology online).Additionally, at screening and at days 3, 14, 28 and 42, a painassessment and blood testing for cytokine levels will be performed inorder to assess physical signs and symptoms of FM as well as thebiochemical effects of the vaccination protocol, respectively.

In order to document daily the intensity of pain, adverse events andconcomitant medications, patients will use a standardized diary and willrecord daily the parameters mentioned. In addition, changes infunctional symptoms will be documented at start of treatment, as well ason day 3, day 14, day 28 and at the end of treatment (day 42). Adverseevents will be assessed during the active treatment period.

To evaluate pain, the pain score, a visual analogue scale and clinicalexamination of tender points will be used. The pain score ranges from 0to 120, measuring the pain intensity in 24 body regions applied to thefollowing rating scale: 0=no pain, 1=mild pain, 2=moderate pain,3=moderately severe pain, 4=severe pain, 5=most ever pain. Theassessment of each body region will be done by the patients themselves;the total score will be calculated as the sum of the regional scores.

The visual analogue scale is in the form of 100-mm-line orientedhorizontally with one end=0, indicating “no pain” and the other end=100,indicating “worst pain”. The patients are asked to place a markcorresponding to their perception of their present pain intensity.

In addition to the documented effects during the active treatment phase,a follow-up of the patients will be performed for six months in order toevaluate the duration of the clinical response (as defined by a 35% orhigher reduction in individual pain score/baseline versus end oftreatment).

Cytokine Assay

In order to measure cytokine levels (i.e., IL-2 and IFN-gamma), atscreening and at days 3, 14, 28 and 42, twenty-30 mL of blood will bedrawn from each patient from each cohort in 7-mL tubes containing 0.081mL of 15% K₃-EDTA solution (BD Vacutainer®, BD, Franklin Lakes, N.J.) byvenipuncture. Blood will be transferred to 50-mL tubes and diluted 1:1in GIBCO® Hank's balanced salt solution (HBSS, Invitrogen, Carlsbad,Calif.). The resulting solution will be layered on top of 15 mL Ficoll(Histopaque®-1077, Sigma-Aldrich, St. Louis, Mo.) in 50-mL tubes andcentrifuged at 1800 rpm for 20 minutes. The top layer will aspirated anddiscarded and the interphase containing PMBC will be harvested andtransferred into a new 50-mL tube. The tube will be filled with HBSS andthe contents mixed by gentle rocking. Cells will be collected bycentrifugation at 2000 rpm for 10 minutes and resuspended in 10 mLcomplete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1%penicillin-streptomycin mixture and 1% L-glutamine solution (Invitrogen,Carlsbad, Calif.). Cell viability and count will be determined usingTrypan blue exclusion assay.

Cytokine and chemokine concentrations in plasma will be measured bymultiplex immunoassay based on Luminex xMAP™ bead array technology andBio-Plex 200 fluorescence bead reader (BioRad Laboratories, Hercules,Calif.). Three panels of antibody-conjugated beads for measuring humaninflammatory cytokines (GM-CSF, IL-1β, IL-6, IL-8, TNF-α), Th1/Th2cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-10) and chemokines (MIP-1α,MIP-1β, MCP-1, Eotaxin, RANTES) (BioSource, Camarillo, Calif.) will beused in the assay according to the manufacturer instructions. Otherpanels that can be included are IL-1RA, IL-2R, IL-7, IL-12 (p40/p70),IL-13, IL-15, IL-17, IFN-, IP-10, MIG, VEGF, EGF, FGF-basic, and HGF(InVitrogen) or IL-1RA, IL-7, IL-9, IL-12, IL13, IL-15, IL-17,FGF-basic, G-CSF, IP-10, VEGF, PDGF-BB (BioRad).

Concentration values will be transferred to Microsoft Office Excel 2003software (Microsoft Corporation, Redmond, Wash.) and means as well asstandard deviations will be calculated for each cytokine concentration.Cytokine concentrations in patients from cohorts 2 and 3 will becompared to those of cohort 1 by using Student's t-test. The confidencelevel will be set at 5%. In addition, analysis of a potentialcorrelation between cytokine induction (i.e., IL-2 and/or IFN-gamma) andmodification of physical/function FM signs & symptoms will be performedusing software and statistical analyses.

Example 3: A Placebo-Controlled Study to Evaluate the Safety andEfficacy of a BCG Vaccine in Treating Chronic Fatigue Syndrome (CFS)

The primary objective of this study will be to evaluate the efficacy ofa BCG vaccine for boosting the immune system of patients suffering fromCFS. Secondary objectives will be to evaluate the safety andtolerability of both of the BCG vaccine as well as investigate localinjection site reactions.

Study Patients and Dosing Schedule

The study will be in the form of a prospective, randomized,double-blind, placebo-controlled, parallel-group study using a BCGvaccine. Male and female patients (over 18 years) who have beenpreviously diagnosed with CFS are included in this trial. The mainexclusion criteria will include pregnant and lactating woman, patientssuffering from other inflammatory rheumatological diseases (such asrheumatoid arthritis or collagenoses), severe neuropathies, clinicallymanifest endocrinopathies, bone diseases, severe cardial, renal orhepatic impairment and acute or chronic infections.

50 Patients will be randomly assigned to one of two study cohorts:placebo (25 patients; cohort 1) or BCG vaccine (25 patients; cohort 2).The duration of treatment will be four weeks over which each patientwill receive (3) intradermal (i.d.) injections. Each dose will beadministered in each patient from one of the two cohorts into the skinoverlying the deltoid muscle with the arm alternated for each dose.Prior to commencement of the study, each patient in each cohort willreceive a placebo injection of borate saline solution (day −3) toprovide an intra-patient placebo control and to allow the patient topractice completion of the diary and assess whether patients are capableof measuring their own in injection site reactions accurately. Patientswho are willing and able to proceed with the study will be injected witha single dose level of placebo (borate saline-solution, cohort 1) or BCG(cohort 2) on (3) subsequent occasions. Doses of placebo or BCG will beadministered over a 4-week period on days 0, 14 and 28 (with up to 2days variation in the dosing interval). For cohort 1, a standard volumeof 0.1 ml of borate saline-solution will be injected. For cohort 2, astandard volume of 0.1 ml of a suspension containing BCG at theconcentration of 10 mg/ml will be injected.

Before, on each dosing day and at the end of the treatment phase (i.e.,at screening and at days 3, 14, 28 and 42), routine safety assessmentswill be performed using physical examinations, urinalysis,electrocardiograms and hematological and biochemical blood tests.Further, at days 0, 3, 14, 28 and 42, local tolerability will beassessed using standardized techniques (measurements at injection site)by a study physician or research nurse and the intensity of eachinjection site reaction will be scored with reference to a VaccineToxicology Rating Scale (available at Annals of Oncology online).Additionally, at screening and at days 3, 14, 28 and 42, a physicalexamination, a pain assessment and full medical write-up will beperformed in order to assess physical signs and symptoms of CFS as wellas the biochemical effects of the vaccination protocol, respectively.

In order to document daily the intensity of pain, adverse events andconcomitant medications, patients will use a standardized diary and willrecord daily the parameters mentioned. In addition, changes infunctional symptoms will be documented at start of treatment, as well ason day 3, day 14, day 28 and at the end of treatment (day 42). Adverseevents will be assessed during the active treatment period.

To evaluate pain, the pain score, a visual analogue scale and clinicalexamination of tender points will be used. The pain score ranges from 0to 120, measuring the pain intensity in 24 body regions applied to thefollowing rating scale: 0=no pain, 1=mild pain, 2=moderate pain,3=moderately severe pain, 4=severe pain, 5=most ever pain. Theassessment of each body region will be done by the patients themselves;the total score will be calculated as the sum of the regional scores.

The visual analogue scale is in the form of 100-mm-line orientedhorizontally with one end=0, indicating “no pain” and the other end=100,indicating “worst pain”. The patients are asked to place a markcorresponding to their perception of their present pain intensity.

In addition to the documented effects during the active treatment phase,a follow-up of the patients will be performed for six months in order toevaluate the duration of the clinical response (as defined by a 35% orhigher reduction in individual pain score/baseline versus end oftreatment).

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

All publications, patents and patent applications herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. It is not an admission thatany of the information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

While the invention has been described in connection with specificembodiments thereof, the foregoing description has been given forclearness of understanding only and no unnecessary limitations should beunderstood therefrom. It will be understood that the description iscapable of further modifications and this application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure as come within known or customarypractice within the art to which the invention pertains and as may beapplied to the essential features hereinbefore set forth and as followsin the scope of the appended claims. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

What is claimed is:
 1. A method of treating fibromyalgia (FM) in asubject, comprising administering a therapeutically effective amount ofa vaccine to the subject, wherein the vaccine comprises Mycobacteriumaurum.
 2. The method of claim 1, wherein the M. aurum is live-attenuatedor heat-killed.
 3. The method of claim 1, wherein the vaccine inducesone or more epigenetic changes in the genome of the subject.
 4. Themethod of claim 1, wherein the administration of the vaccine alleviates,prevents or reduces at least one sign or symptom of fibromyalgia,wherein the sign or symptom is selected from chronic muscle pain, musclespasms, muscle tightness, moderate or severe fatigue, decreased energy,insomnia, feeling of exhaustion, stiffness upon waking, stiffness afterstaying in one position for too long, difficulty remembering, difficultyconcentrating, difficulty performing simple mental tasks (“fibro fog”),abdominal pain, bloating, nausea, constipation alternating with diarrhea(irritable bowel syndrome), tension or migraine headaches, jaw andfacial tenderness, sensitivity to one or more of odors, noise, brightlights, medications, certain foods, and cold, feeling anxious ordepressed, numbness or tingling in the face, arms, hands, legs, or feet,increase in urinary urgency or frequency (irritable bladder), reducedtolerance for exercise and muscle pain after exercise, a feeling ofswelling (without actual swelling) in the hands and feet or anycombination of symptoms thereof.
 5. The method of claim 1, wherein theadministration of the vaccine elevates or increases immune systemactivity of the subject.
 6. The method of claim 5, wherein the elevationor increase in immune system function is evidenced by the production ofT_(H)1 cytokines, upregulation of granzyme B or both.
 7. The method ofclaim 1, further comprising prior to the administering step: determiningdysfunction in peripheral blood mononuclear cells (PBMCs), wherein thedetermining the dysfunction comprises: (a) stimulating a samplecomprising PBMCs obtained from the subject; and (b) measuring expressionlevels of at least four cytokines selected from the group consisting ofIL-2, IL-4, IL5, IL6, IL8, IL 10, IFN-γ, RANTES, MCP-1, MIP-α and MIP-βin a PBMC sample obtained from the subject; comparing the expressionlevels of the at least four cytokines in the PBMC sample obtained fromthe subject to the expression levels of the at least four cytokines in acontrol sample of PBMCs; and diagnosing the subject with FM ifdysfunction in PBMCs is detected in the PBMCs obtained from the subject,wherein the dysfunction is indicated by altered expression levels of theat least four cytokines in the PBMC sample obtained from the subject ascompared to the expression levels of the at least four cytokines in thecontrol PBMC sample.
 8. The method of claim 7, wherein the PBMCs areisolated from plasma of a blood sample obtained from the subject.
 9. Themethod of claim 7, wherein the PBMCs are stimulated with one or moremitogens.
 10. The method of claim 7, wherein the determined expressionlevel for each of the at least four cytokines is a protein expressionlevel, wherein the protein expression level is determined using anantibody bead-based capture assay that comprises beads derivatized withantibodies specific to the at least four cytokines.
 11. A method ofelevating or increasing a subject's immune system function comprisingadministering a therapeutically effective amount of a vaccine to thesubject, wherein the subject suffers from or is suspected of sufferingfrom fibromyalgia and wherein the vaccine comprises Mycobacterium aurum(M. aurum).
 12. The method of claim 11, wherein the elevation orincrease in immune system function is evidenced by the production ofT_(H)1 cytokines, upregulation of granzyme B or both.
 13. The method ofclaim 11, wherein the elevation or increase in immune system function isevidenced by a reduction or alleviation of at least one sign or symptomof fibromyalgia.
 14. The method of claim 13, wherein the sign or symptomof fibromyalgia is selected from chronic muscle pain, muscle spasms,muscle tightness, moderate or severe fatigue, decreased energy,insomnia, a feeling of exhaustion, stiffness upon waking, stiffnessafter staying in one position for too long, difficulty remembering,difficulty concentrating, difficulty performing simple mental tasks(“fibro fog”), abdominal pain, bloating, nausea, and constipationalternating with diarrhea (irritable bowel syndrome), tension ormigraine headaches, jaw and facial tenderness, sensitivity to one ormore of odors, noise, bright lights, medications, certain foods, andcold, feeling anxious or depressed, numbness or tingling in the face,arms, hands, legs, or feet, increase in urinary urgency or frequency(irritable bladder), reduced tolerance for exercise and muscle painafter exercise, a feeling of swelling (without actual swelling) in thehands and feet or a combination of symptoms thereof.
 15. The method ofclaim 11, wherein the M. aurum induces one or more epigenetic changes inthe genome of the individual.
 16. The method of claim 11, wherein the M.aurum is live-attenuated or heat-killed.
 17. The method of claim 11,further comprising prior to the administering step: determiningdysfunction in peripheral blood mononuclear cells (PBMCs), wherein thedetermining the dysfunction comprises: (a) stimulating a samplecomprising PBMCs obtained from the subject; and (b) measuring expressionlevels of at least four cytokines selected from the group consisting ofIL-2, IL-4, IL5, IL6, IL8, IL 10, IFN-γ, RANTES, MCP-1, MIP-α and MIP-βin a PBMC sample obtained from the subject; comparing the expressionlevels of the at least four cytokines in the PBMC sample obtained fromthe subject to the expression levels of the at least four cytokines in acontrol sample of PBMCs; and diagnosing the subject with FM ifdysfunction in PBMCs is detected in the PBMCs obtained from the subject,wherein the dysfunction is indicated by altered expression levels of theat least four cytokines in the PBMC sample obtained from the subject ascompared to the expression levels of the at least four cytokines in thecontrol PBMC sample.
 18. The method of claim 17, wherein the PBMCs areisolated from plasma of a blood sample obtained from the subject. 19.The method of claim 17, wherein the PBMCs are stimulated with one ormore mitogens.
 20. The method of claim 17, wherein the determinedexpression level for each of the at least four cytokines is a proteinexpression level, wherein the protein expression level is determinedusing an antibody bead-based capture assay that comprises beadsderivatized with antibodies specific to the at least four cytokines.